U.S. patent application number 10/208374 was filed with the patent office on 2003-03-13 for method of reducing injury to mammalian cells.
Invention is credited to Tymianski, Michael.
Application Number | 20030050243 10/208374 |
Document ID | / |
Family ID | 46280960 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030050243 |
Kind Code |
A1 |
Tymianski, Michael |
March 13, 2003 |
Method of reducing injury to mammalian cells
Abstract
A method of inhibiting the binding between N-methyl-D-aspartate
receptors and neuronal proteins in a neuron the method comprising
administering to the neuron an effective inhibiting amount of a
peptide replacement agent for the NMDA receptor or neuronal protein
interaction domain that effect said inhibition of the NMDA receptor
neuronal protein. The method is of value in reducing the damaging
effect of injury to mammalian cells. Postsynaptic density-95
protein (PSD-95) couples neuronal N-methyl-D-aspartate receptors
(NMDARs) to pathways mediating excitotoxicity and ischemic brain
damage. This coupling was disrupted by transducing neurons with
peptides that bind to modular domains on either side of the
PSD-95/NMDAR interaction complex. This treatment attenuated
downstream NMDAR signaling without blocking NMDAR activity,
protected cultured cortical neurons from excitotoxic insults and
dramatically reduced cerebral infarction volume in rats subjected
to transient focal cerebral ischemia. The treatment was effective
when applied either before, or one hour after, the onset of
excitotoxicity in vitro and cerebral ischemia in vivo. This
approach prevents negative consequences associated with blocking
NMDAR activity and constitutes practical therapy for stroke.
Inventors: |
Tymianski, Michael;
(Toronto, CA) |
Correspondence
Address: |
BELL, BOYD & LLOYD LLC
P.O. Box 1135
Chicago
IL
60690-1135
US
|
Family ID: |
46280960 |
Appl. No.: |
10/208374 |
Filed: |
July 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10208374 |
Jul 30, 2002 |
|
|
|
09584555 |
May 31, 2000 |
|
|
|
Current U.S.
Class: |
514/8.3 ;
514/15.1; 514/17.3 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 38/08 20130101; A61P 9/00 20180101; A61P 25/00 20180101; A61P
9/10 20180101 |
Class at
Publication: |
514/12 ;
514/15 |
International
Class: |
A61K 038/17; A61K
038/08; A61K 038/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 1999 |
CA |
2,273,622 |
Claims
I claim:
1. A method of inhibiting the binding between N-methyl-D-aspartate
receptors and neuronal proteins in a neuron in vivo said method
comprising administering to said neuron an effective inhibiting
amount of a peptide replacement agent for a NMDA receptor or
neuronal protein interaction domain to effect inhibition of the
NMDA receptor neuronal protein.
2. A method as defined in claim 1 wherein said neuron is
damaged.
3. A method of reducing the damaging effect of ischemia or
traumatic injury to the brain or spinal chord in a mammal, said
method comprising treating said mammal with a non-toxic,
damage-reducing, effective amount of a peptide replacement agent
for the NMDA receptor or neuronal protein interaction domain to
effect said inhibition of the NMDA receptor neuronal protein.
4. A method as defined in claim 3 wherein said mammal is under the
influence of neuronal cell damage.
5. A method as defined in claim 1 wherein said NMDA receptor is
bindable with proteins containing PDZ domains.
6. A method as defined in claim 5 wherein said protein containing
PDZ domains is PSD-95.
7. A method as defined in claim 5 wherein said protein containing
PDZ domains is PSD-93.
8. A method as defined in claim 5 wherein said protein containing
PDZ domains is SAP102.
9. A method as defined in claim 5 wherein said protein containing
PDZ domains is SAP97.
10. A method as defined in claim 1 wherein said protein containing
PDZ domains is a tSXV-containing peptide.
11. A method as defined in claim 10 wherein said agent is KLSSIESDV
(SEQ ID NO: 1).
12. A method as defined in claim 1 wherein said neuronal protein is
bindable with excitatory amino acid receptors
13. A method as defined in claim 1 wherein said neuronal protein is
bindable with NMDA receptors
14. A method as defined in claim 1 wherein said neuronal protein is
bindable with AMPA receptors
15. A method as defined in claim 1 wherein said neuronal protein is
bindable with metabotropic glutamate receptors
16. A method as defined in claim 1 wherein said agent is a peptide
encoding a PDZ-binding domain of a protein containing PDZ
domains.
17. A method as defined in claim 16 wherein said agent is residues
65-248 of PSD-95.
18. A method of controlling the concentration of
Ca.sup.2+-dependent signaling molecules in the vicinity of ion
channel pores of cells in vivo to prevent the diffusion of toxic
amounts of said Ca.sup.2+ influx to prevent the triggering of
neurotoxic phenomena, said method comprising administering an
effective, non-toxic amount of a peptide replacement agent for the
NMDA receptor or neuronal protein interaction domain that effect
said inhibition of the NMDA receptor neuronal protein.
19. A method as defined in claim 18 wherein said agent is a
tSXV-containing peptide.
20. A method as defined in claim 19 wherein said agent is KLSSIESDV
(SEQ ID NO: 1).
21. A method as defined in claim 18 wherein said agent is a peptide
encoding a PDZ-binding domain of a protein containing PDZ
domains.
22. A method as defined in claim 21 wherein said agent is residues
65-248 of PSD-95.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part application of
Ser. No. 09/584,555, filed May 31, 2000.
FIELD OF THE INVENTION
[0002] This invention relates to methods of reducing the damaging
effect of an injury to mammalian cells by treatment with compounds
which reduce the binding between N-methyl-D-aspartate receptors and
neuronal proteins; pharmaceutical compositions comprising said
compounds and methods for the preparation of said pharmaceutical
compositions.
BACKGROUND TO THE INVENTION
[0003] Ischemic or traumatic injuries to the brain or spinal cord
often produce irreversible damage to central nervous system (CNS)
neurons and to their processes. These injuries are major problems
to society as they occur frequently, the damage is often severe,
and at present there are still no effective pharmacological
treatments for acute CNS injuries. Clinically, ischemic cerebral
stroke or spinal cord injuries manifest themselves as acute
deteriorations in neurological capacity ranging from small focal
defects, to catastrophic global dysfunction, to death. It is
currently felt that the final magnitude of the deficit is dictated
by the nature and extent of the primary physical insult, and by a
time-dependent sequence of evolving secondary phenomena which cause
further neuronal death. Thus, there exists a theoretical
time-window, of uncertain duration, in which a timely intervention
might interrupt the events causing delayed neurotoxicity. However,
little is known about the cellular mechanisms triggering and
maintaining the processes of ischemic or traumatic neuronal death,
making it difficult to devise practical preventative strategies.
Consequently, there are currently no clinically useful
pharmacological treatments for cerebral stroke or spinal cord
injury.
[0004] In vivo, a local reduction in CNS tissue perfusion mediates
neuronal death in both hypoxic and traumatic CNS injuries. Local
hypoperfusion is usually caused by a physical disruption of the
local vasculature, vessel thrombosis, vasospasm, or luminal
occlusion by an embolic mass. Regardless of its etiology, the
resulting ischemia is believed to damage susceptible neurons by
impacting adversely on a variety of cellular homeostatic
mechanisms. Although the nature of the exact disturbances is poorly
understood, a feature common to many experimental models of
neuronal injury is a rise in free intracellular calcium
concentration ([Ca.sup.2+]i). Neurons possess multiple mechanisms
to confine [Ca.sup.2+].sub.i to the low levels, about 100 nM
necessary for the physiological function. It is widely believed
that a prolonged rise in [Ca.sup.2+].sub.i deregulates
tightly-controlled Ca.sup.2+-dependent processes, causing them to
yield excessive reaction products, to activate normally quiescent
enzymatic pathways, or to inactivate regulatory cytoprotective
mechanisms. This, in-turn, results in the creation of
experimentally observable measures of cell destruction, such as
lipolysis, proteolysis, cytoskeletal breakdown, pH alterations and
free radical formation.
[0005] The classical approach to preventing Ca.sup.2+ neurotoxicity
has been through pharmacological blockade of Ca.sup.2+ entry
through Ca.sup.2+ channels and/or of excitatory amino acid
(EAA)--gated channels. Variations on this strategy often lessen
EAA-induced or anoxic cell death in vitro, lending credence to the
Ca.sup.2+-neurotoxicity hypothesis. However, a variety of Ca.sup.2+
channel- and EAA-antagonists fail to protect against neuronal
injury in vivo, particularly in experimental Spinal Cord Injury
(SCI), head injury and global cerebral ischemia. It is unknown
whether this is due to insufficient drug concentrations,
inappropriate Ca.sup.2+ influx blockade, or to a contribution from
non-Ca.sup.2+ dependent neurotoxic processes. It is likely that
Ca.sup.2+ neurotoxicity is triggered through different pathways in
different CNS neuron types. Hence, successful Ca.sup.2+-blockade
would require a polypharmaceutical approach.
[0006] As a result of investigations, I have discovered methods of
reducing the damaging effect of an injury to mammalian cells by
treatment with compounds to reduce the binding between
N-methyl-D-aspartate (NMDA) receptors and neuronal proteins.
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SUMMARY OF THE INVENTION
[0046] I have found that postsynaptic density-95 protein (PSD-95)
couples neuronal N-methyl-D-aspartate receptors (NMDARs) to
pathways mediating excitotoxicity and ischemic brain damage. This
coupling was disrupted by transducing neurons with peptides that
bind to modular domains on either side of the PSD-95/NMDAR
interaction complex. This treatment attenuated downstream NMDAR
signaling without blocking NMDAR activity, protected cultured
cortical neurons from excitotoxic insults and dramatically reduced
cerebral infarction volume in rats subjected to transient focal
cerebral ischemia. The treatment was effective when applied either
before, or one hour after, the onset of excitotoxicity in vitro and
cerebral ischemia in vivo. This approach may prevent negative
consequences associated with blocking NMDAR activity and constitute
a practical therapy for stroke.
[0047] It is an object of the present invention to provide in its
broadest aspect a method of reducing the damaging effect of an
injury to mammalian cells.
[0048] In a preferred object, the invention provides pharmaceutical
compositions for use in treating mammals to reduce the damaging
effect of an injury to mammalian tissue.
[0049] The present invention is based on the discovery of a
neuroprotective effect against excitotoxic and ischemic injury by
inhibiting the binding between N-methyl-D-aspartate (NMDA)
receptors and neuronal proteins in a neuron.
[0050] Accordingly, in one aspect the invention provides a method
of inhibiting the binding between N-methyl-D-aspartate receptors
and neuronal proteins in a neuron said method comprising
administering to said neuron an effective inhibiting amount of a
peptide replacement agent for the NMDA receptor interaction domain
to effect said inhibition of the interaction with the neuronal
protein.
[0051] In a further aspect, the invention provides a method of
inhibiting the binding between N-methyl-D-aspartate receptors and
neuronal proteins in a neuron said method comprising administering
to said neuron an effective inhibiting amount of a peptide
replacement agent for the neuronal protein interaction domain to
effect said inhibition of the interaction with the NMDA
receptor.
[0052] In a further aspect, the invention provides a method of
reducing the damaging effect of ischemia or traumatic injury to the
brain or spinal cord in a mammal, said method comprising treating
said mammal with a non-toxic, damage-reducing, effective amount of
a peptide replacement agent for the NMDA receptor or neuronal
protein interaction domains that inhibit the NMDA receptor neuronal
protein interaction.
[0053] Damage to neurons in this specification is meant anoxia,
ischemia, excitotoxicity, lack of neurotrophic support,
disconnection, and mechanical injury.
[0054] The NMDA agent is, preferably, bindable with proteins
containing PDZ domains, and most preferably, is selected from
postsynaptic density-95 proteins, PSD-95, PSD-93 and SAP102.
[0055] I have found that the replacement agent is a tSXV-containing
peptide, preferably KLSSIESDV (SEQ. ID NO: 1).
[0056] The neuronal protein agent is, preferably, bindable with
excitatory amino acid receptors, and most preferably, is selected
from NMDA receptor subunits NR1 and NR2.
[0057] I have found that the replacement agent is a PDZ2-domain
containing polypeptide, preferably corresponding to residues 65-248
of PSD-95, encoding the first and second PDZ domains (PDZ1-2) of
PSD-95.
[0058] In a yet further aspect the invention provides a
pharmaceutical composition comprising a peptide replacement agent
for the NMDA receptor or neuronal protein interaction domains that
inhibit the NMDA receptor neuronal protein interaction in a mixture
with a pharmaceutically acceptable carrier when used for reducing
the damaging effect of an ischemic or traumatic injury to the brain
or spinal chord of a mammal; preferably further comprising the
cell-membrane transduction domain of the human immunodeficiency
virus type 1 (HIV-1) Tat protein (YGRKKRRQRRR (SEQ ID No: 2); Tat),
or the antennapedia internalisation peptide.
[0059] In a most preferred aspect, the invention provides a
pharmaceutical composition comprising the peptide KLSSIESDV (SEQ ID
NO: 1) or residues 65-248 of PSD-95, encoding the first and second
PDZ domains (PDZ1-2) of PSD-95.
[0060] In a further aspect, the invention provides a method of
inhibiting the binding between NMDA receptors and neuronal proteins
in a neuron, said method comprising administering to said neuron an
effective inhibiting amount of an antisense DNA to prevent
expression of said neuronal proteins to effect inhibition of said
binding. Preferably, this aspect provides a method wherein said
antisense DNA reduces the expression of a protein containing PDZ
domains bindable to said NMDA receptor. More preferably, the
protein containing PDZ domains is selected from PSD-95, PSD-93 and
SAP102.
[0061] In the mammalian nervous system, the efficiency by which
N-methyl-D-aspartate receptor (NMDAR) activity triggers
intracellular signaling pathways governs neuronal plasticity,
development, senescence and disease. I have studied excitotoxic
NMDAR signaling by suppressing the expression of the NMDAR
scaffolding protein PSD-95. In cultured cortical neurons, this
selectively attenuated NMDAR excitotoxicity, but not excitotoxicity
by other glutamate or Ca.sup.2+ channels. NMDAR function was
unaffected, as receptor expression, while NMDA-currents and
.sup.45Ca loading via NMDARs were unchanged. Suppressing PSD-95
selectively blocked Ca.sup.2+-activated nitric oxide production by
NMDARs, but not by other pathways, without affecting neuronal
nitric oxide synthase (nNOS) expression or function. Thus, PSD-95
is required for the efficient coupling of NMDAR activity to nitric
oxide toxicity and imparts specificity to excitotoxic Ca.sup.2+
signaling.
[0062] It is known that calcium influx through NMDARs plays key
roles in mediating synaptic transmission, neuronal development, and
plasticity (1). In excess, Ca influx triggers excitotoxicity (2), a
process that damages neurons in neurological disorders that include
stroke, epilepsy, and chronic neurodegenerative conditions (3).
Rapid Ca.sup.2+-dependent neurotoxicity is triggered most
efficiently when Ca.sup.2+ influx occurs through NMDARs, and cannot
be reproduced by loading neurons with equivalent quantities of
Ca.sup.2+ through non-NMDARs or voltage-sensitive Ca.sup.2+
channels (VSCCs) (4). This observation suggests that Ca.sup.2+
influx through NMDAR channels is functionally coupled to neurotoxic
signaling pathways.
[0063] Without being bound by theory, I believe that lethal
Ca.sup.2+ signaling by NMDARs is determined by the molecules with
which they physically interact. The NR2 NMDAR subunits, through
their intracellular C-terminal domains, bind to PSD-95/SAP90 (5),
chapsyn-110/PSD-93, and other members of the membrane-associated
guanylate kinase (MAGUK) family (6). NMDAR-bound MAGUKs are
generally distinct from those associated with non-NMDARs (7). I
have found that the preferential activation of neurotoxic Ca.sup.2+
signals by NMDARs is determined by the distinctiveness of
NMDAR-bound MAGUKs, or of the intracellular proteins that they
bind. PSD-95 is a submembrane scaffolding molecule that binds and
clusters NMDARs preferentially and, through additional
protein-protein interactions, may link them to intracellular
signaling molecules (8). Perturbing PSD-95 would impact on
neurotoxic Ca.sup.2+ signaling through NMDARs.
[0064] Thus, protein-protein interactions govern the signals
involved in cell growth, differentiation, and intercellular
communication through dynamic associations between modular protein
domains and their cognate binding partners (20). At excitatory
synapses of central neurons, ionotropic glutamate receptors are
organized into multi-protein signaling complexes within the
post-synaptic density (PSD) (21). A prominent organizing protein
within the PSD is PSD-95, a member of the membrane-associated
guanylate kinase (MAGUK) family. PSD-95 contains multiple domains
that couple transmembrane proteins such as the N-methyl-D-aspartate
subtype of glutamate receptors (NMDAR) to a variety of
intracellular signaling enzymes (21,22). Through its second PDZ
domain (PDZ2), PSD-95 binds both the NMDAR 2B subunit (NR2B) and
neuronal nitric oxide synthase (nNOS) (22). This interaction
couples NMDAR activity to the production of nitric oxide (NO), a
signaling molecule that mediates NMDAR-dependent excitotoxicity
(23). Research has shown that NMDAR function is unaffected by
genetically disrupting PSD-95 in vivo (24) or by suppressing its
expression in vitro (25). Nonetheless, PSD-95 deletion dissociates
NMDAR activity from NO production and suppresses NMDAR-dependent
excitotoxicity.
[0065] Although NMDARs play an important neurotoxic role in
hypoxic/ischemic brain injury (26), blocking NMDAR function may be
deleterious in animals and humans (27-29). Targeting PSD-95 protein
therefore represents an alternative therapeutic approach for
diseases that involve excitotoxicity that may circumvent the
negative consequences of blocking NMDAR function. However, mutation
or suppression of PSD-95 is impractical as a therapy for brain
injury and cannot be applied after an injury has occurred.
Therefore, rather than alter PSD-95 expression, I questioned
whether interfering with the NMDAR/PSD-95 interaction could
suppress excitotoxicity in vitro and ischemic brain damage in
vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] In order that the invention may be better understood
preferred embodiments will now be described by way of example only
with reference to the accompanying drawings wherein:
[0067] FIG. 1a is an immunublot;
[0068] FIG. 1b is a bar chart providing densitometric analysis of
PSD-95 expression;
[0069] FIG. 1c represents representative phase contrast and
propidium fluorescence images;
[0070] FIG. 1d is a bar chart of NMDA concentration against
fraction of dead cells;
[0071] FIG. 1e is a bar chart of NMDA concentration against Calcium
accumulation.
[0072] FIGS. 2a1-b2 represent bar charts of selective activations
of AMPA/Kainate receptors with Kainate (2a1 and 2-a2); and loadings
with Vscc's (2-b1) and calcium loading (2-b2).
[0073] FIGS. 3a-d represent immunoblots (3a); NMDA dose-response
curves (3b); NMDA current density measurements (3c); and
current/time graph (3d) dialyzed with hucifer yellow;
[0074] FIG. 4 bar charts (4a; 4c-4f) and immunoblot 4b of effect on
nNOS expression in cultures are hereinafter better described and
explained;
[0075] FIG. 5. (A) Shows the hypothesis: The NMDAR/PSD-95 complex
may be dissociated by peptides encoding either to the C-terminus of
NR2 or the second PDZ domains of PSD-95 (B) Fluorescence of
cultures treated with Tat-38-48-dansyl and Tat-NR2B9c dansyl. (C)
Time course fluorescence after Tat-NR2B9c-dansyl application (D)
Effect of peptides on co-immunoprecipitation of PSD-95 with
NR2B
[0076] FIG. 6. Effect of Tat-NR2B9c on (A-C) electrophysiological
function of neurons (D) NMDA-evoked .sup.45Ca.sup.2+ uptake in
cortical cultures. (E) NMDA-evoked cGMP production in cortical
cultures. (F) NMDA-evoked excitotoxicity in cortical cultures.
[0077] FIG. 7. (A) Detection of Tat-NR2B9c-dansyl in the mouse
brain 1 h after intraperitoneal injection (B) Composite
neurological scores (see text) during and 24 h after MCAo. (C)
Effect of Pre-treatment with Tat-NR2B9c on (i) total infarct area
and volume (inset), and (ii) cortical infarct area and volume
(inset) after transient MCAo.
[0078] FIG. 8. (A) Neuroprotective effects of post-treatment in
cultured cortical neurons post-treated with Tat-NR2B9c or
pTat-PDZ1-2 (B) Composite neurological scores (see text) during and
24 h after MCAo. (C) Effect of post-treatment with Tat-NR2B9c on
(i) total infarct area and volume (inset), and (ii) cortical
infarct area and volume (inset) after transient MCAo.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0079] Methods:
[0080] Cultured cortical neurons were prepared by standard
techniques (4,9) and switched to serum-free media at 24 h
[Neurobasal with B27 supplement (Gibco)]. The AS ODN corresponded
to nucleotides 435-449 of mouse PSD-95/SAP90 mRNA (GeneBank Acc.
No. D50621). Filter-sterilized phosphodiester AS SE, and MS ODNs (5
.mu.M) were added in culture medium during feedings at 4,6,8 and 10
days after plating. Cultures were used for all experiments (FIGS.
1-4) on day 12. ODN sequences exhibited no similarity to any other
known mammalian genes (BLAST search (10)).
[0081] Immunoblotting was done as described in ref. "26". Tissue
was harvested and pooled from 2 cultures/lane. The blotted proteins
were probed using a monoclonal anti-PSD-95 mouse IgG1 (Transduction
Labs, 1:250 dilution), polyclonal anti PSD-93 (1:1000 dilution) and
anti SAP-102 (1:2000 dilution) rabbit serum antibodies (Synaptic
Systems GmbH), a monoclonal anti NR1 mouse IgG2a (PharMingen
Canada, 1:1000 dilution) or a monoclonal anti nNOS (NOS type I)
mouse IgG2a (Transduction Labs, 1:2500 dilution). Secondary
antibodies were sheep anti-mouse, or donkey anti-rabbit Ig
conjugated to horseradish peroxidase (Amersham). Immunoblots for
PSD-95 were obtained for all experiments (FIGS. 1-4) from sister
cultures, and all gels quantified using an imaging densitometer
(Bio-Rad GS-670).
[0082] cGMP determinations were performed 10 min after challenging
the cultures with NMDA, kainate, or high-K (FIGS. 4c-e) with the
Biotrak cGMP enzymeimmunoassay system according to the kit
manufacturer's instructions (Amersham). Staining for NADPH
diaphorase (FIG. 4b) was done as described in ref. 12.
[0083] Electrophysiology. Whole cell patch-clamp recordings in the
cultured neurons were performed and analyzed as described in ref.
13. During each experiment a voltage step of -10 mV was applied
from holding potential and the cell capacitance was calculated by
integrating the capacitative transient. The extracellular solution
contained (in mM): 140 NaCl, 5.4 KCl, 1.3 CaCl.sub.2, 25 HEPES, 33
glucose, 0.01 glycine, and 0.001 tetrodotoxin (pH=7.3-7.4, 320-335
mOsm). A multi-barrel perfusion system was employed to rapidly
exchange NMDA containing solutions. The pipette solution contained
(in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2 tetraethylammonium
chloride (TEA), 1 CaCl.sub.2, 4 MgATP, pH 7.3 at 300 mOsm. Lucifer
yellow (LY; 0.5% w/v) was included in the pipette for experiments
in FIG. 3d.
[0084] Excitotoxicity and Ca.sup.2+ accumulation measurements were
performed identically to the methods described and validated in
refs. 4 and 14. We used measurements of propidium iodide
fluorescence as an index of cell death, and of radiolabelled
.sup.45Ca.sup.2+ accumulation for Ca.sup.2+ load determinations in
sister cultures on the same day. Experimental solutions were as
previously described (4). Ca.sup.2+ influx was pharmacologically
channeled through distinct pathways as follows: To NMDARs by
applying NMDA (.times.60 min) in the presence of both CNQX
(Research Biochemicals Inc) and nimodipine (Miles Pharmaceuticals),
to non-NMDARs by applying kainic acid (.times.60 min or 24 h) in
the presence of both MK-801 (RBI) and nimodipine, and to VSCCs
using 50 mM K+ solution (.times.60 min) containing 10 mM Ca.sup.2+
and S(-)-Bay K 8644, an L-type channel agonist (300-500 nM; RBI),
MK-801 and CNQX. Antagonist concentrations were (in .mu.M): MK-801
10, CNQX 10, nimodipine 2. All three antagonists were added after
the 60 min agonist applications for the remainder of all
experiments (24 h). A validation of this approach in isolating
Ca.sup.2+ influx to the desired pathway in our cortical cultures
has been published (4).
[0085] Whole cell patch-clamp recordings in the cultured neurons
were performed and analyzed as described in Z. Xiong, W. Lu, J. F.
MacDonald, Proc Natl Acad Sci USA 94, 7012 (1997). During each
experiment a voltage step of -10 mV was applied from holding
potential and the cell capacitance was calculated by integrating
the capacitative transient. The extracellular solution contained
(in mM): 140 NaCl, 5.4 KCl, 1.3 CaCl.sub.2, 25 HEPES, 33 glucose,
0.01 glycine, and 0.001 tetrodotoxin (pH=7.3-7.4, 320-335 mOsm). A
multi-barrel perfusion system was employed to rapidly exchange NMDA
containing solutions. The pipette solution contained (in mM): 140
CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2 tetraethylammonium chloride
(TEA), 1 CaCl.sub.2, 4 MgATP, pH 7.3 at 300 mOsm. Lucifer yellow
(LY; 0.5% w/v) was included in the pipette for experiments in FIG.
3D.
[0086] Data analysis: data in all figures were analyzed by ANOVA,
with a post-hoc Student's t-test using the Bonferroni correction
for multiple comparisons. All means are presented with their
standard errors.
[0087] In greater detail:
[0088] FIG. 1, shows increased resilience of PSD-95 deficient
neurons to NMDA toxicity in spite of Ca.sup.2+ loading. A.
Immunoblot showing representative effects of sham (SH) washes, and
PSD-95 AS, SE and MS ODNs, on PSD-95 expression. PC: positive
control tissue from purified rat brain cell membranes. Asterisk:
non-specific band produced by the secondary antibody, useful to
control for protein loading and blot exposure times. B.
Densitometric analysis of PSD-95 expression pooled from N
experiments. Asterisk: different from other groups, one-way ANOVA,
F=102, p<0.0001. ODNs were used at 5 .mu.M except where
indicated (AS 2 .mu.M). C. Representative phase contrast and
propidium iodide fluorescence images of PSD-95 deficient (AS) and
control (SE) cultures 24 h after a 60 min challenge with 30 .mu.M
NMDA. Scale bar: 100 .mu.m. D. Decreased NMDA toxicity at 24 h in
PSD-95 deficient neurons following selective NMDAR
activation.times.60 min (n=16 cultures/bar pooled from N=4 separate
experiments). Asterisk: differences from SE, MS and SH (Bonferroni
t-test, p<0.005). Death is expressed as the fraction of dead
cells produced by 100 .mu.M NMDA in sham-ODN-treated controls
(validated in 4,14). E. No effect of PSD-95 deficiency on
NMDAR-mediated Ca.sup.2+ loading (n=12/bar, N=3; reported as the
fraction of .sup.45Ca.sup.2+ accumulation achievable over 60 min in
the sham controls by 100 .mu.M NMDA, which maximally loads the
cells with calcium (4).
[0089] FIG. 2, shows thatPSD-95 deficiency does not affect toxicity
and Ca.sup.2+ loading produced by activating non-NMDARs and
Ca.sup.2+ channels. Cultures were treated with SH washes or AS or
SE ODNs as in FIG. 1. A. Selective activation of AMPA/kainate
receptors with kainate in MK-801 (10 .mu.M) and nimodipine (NIM; 2
.mu.M) produces toxicity over 24 h (A1) irrespective of PSD-95
deficiency, with minimal .sup.45Ca.sup.2+ loading (A2). B.
Selective activation of VSCCs produces little toxicity (B1), but
significant .sup.45Ca.sup.2+ loading (B2) that is also insensitive
to PSD-95 deficiency. n=4 cultures/bar in all experiments.
[0090] FIG. 3, shows that there is no effect of perturbing PSD-95
on receptor function. A. Immunoblots of PSD-95 ODN-treated cultures
probed for PSD-95, NR1, PSD-93, and SAP-102 using specific
antibodies. PC: positive control tissue from purified rat brain
cell membranes. B. NMDA dose-response curves and representative
NMDA currents (inset) obtained with 3-300 .mu.M NMDA. C. NMDA
current density measurements elicited with 300 .mu.M NMDA (AS:
n=18; SE: n=19; SH: n=17; one-way ANOVA F=1.10, p=0.34), and
analysis of NMDA current desensitization. I.sub.ss=steady-state
current; I.sub.peak=peak current. AS: n=15; SE: n=16; SH: n=16
(ANOVA,, F=0.14, p=0.87). Time constants for current decay were AS:
1310.+-.158 ms; SE, 1530.+-.185 ms; SH: 1190.+-.124 ms (ANOVA,
F=1.22, p=0.31). D. Currents elicited with 300 .mu.M NMDA in
neurons dialyzed with LY (insert) and 1 mM tSXV or control
peptide.
[0091] FIG. 4, shows the effect of coupling of NMDAR activation to
nitric oxide signaling by PSD-95. A. L-NAME protects against NMDA
toxicity (n=4, N=2). Asterisk: difference from 0 .mu.M L-NAME
(Bonferroni t-test, p<0.05). B. No effect of SH and of PSD-95 AS
and MS ODNs on nNOS expression in cultures (immunoblot) and on
NADPH diaphorase staining in PSD-95 AS and SE-treated neurons. PC:
positive control tissue from purified rat brain cell membranes. C.
Effect of isolated NMDAR activation on cGMP formation (n=12
cultures/bar pooled from N=3 separate experiments) D,E. Effects of
VSCC activation (n=8/bar, N=2), and AMPA/kainate receptor
activation (n=4/bar, N=1) on cGMP formation. Data in C-E are
expressed as the fraction of cGMP produced in SE-treated cultures
by 100 .mu.M NMDA. Asterisk: differences from both SH and SE
controls (Bonferroni t-test, p<0.0001). F. Sodium nitroprusside
toxicity is similar in PSD-95 AS, SE and SH treated cultures.
[0092] PSD-95 expression was suppressed in cultured cortical
neurons to<10% of control levels, using a 15-mer phosphodiester
antisense (AS) oligodeoxynucleotide (ODN) (FIGS. 1A,B) Sham (SH)
washes, sense (SE) and missense (MS) ODNs (9) had no effect. The
ODNs had no effect on neuronal survivability and morphology as
gauged by viability assays, herein below, and phase-contrast
microscopy (not shown).
[0093] To examine the impact of PSD-95 on NMDAR-triggered
excitotoxicity, ODN-treated cultures were exposed to NMDA (10-100
.mu.M) for 60 min, washed, and either used for .sup.45Ca.sup.2+
accumulation measurements, or observed for a further 23 h.
Ca.sup.2+ influx was isolated to NMDARs by adding antagonists of
non-NMDARs and Ca.sup.2+ channels (4). NMDA toxicity was
significantly reduced in neurons deficient in PSD-95 across a range
of insult severities (FIGS. 1C,D; EC.sub.50: AS: 43.2.+-.4.3; SE:
26.3.+-.3.4, Bonferroni t-test, p<0.005). Concomitantly however,
PSD-95 deficiency had no effect on Ca.sup.2+ loading into
identically treated sister cultures (FIG. 1E). Therefore, PSD-95
deficiency induces resilience to NMDA toxicity despite maintained
Ca.sup.2+ loading.
[0094] I next examined whether the increased resilience to
Ca.sup.2+ loading in PSD-95 deficient neurons was specific to
NMDARs. Non-NMDAR toxicity was produced using kainic acid (30-300
.mu.M), a non-desensitizing AMPA/kainate receptor agonist (15), in
the presence of NMDAR and Ca.sup.2+ channel antagonists (4).
Kainate toxicity was unaffected in PSD-95 deficient in neurons
challenged for either 60 min (not shown) or 24 h (FIG. 2A1).
Non-NMDAR toxicity occurred without significant .sup.45Ca.sup.2+
loading (FIG. 2A2), as>92% of neurons in these cultures express
Ca.sup.2+-impermeable AMPA receptors (4). However, Ca.sup.2+
loading through VSCCs, which is non-toxic (4) (FIG. 2B1), was also
unaffected by PSD-95 deficiency (FIG. 2B2). Thus, suppressing
PSD-95 expression affects neither toxicity nor Ca.sup.2+ fluxes
triggered through pathways other than NMDARs.
[0095] Immunoblot analysis (11) of PSD-95 deficient cultures
revealed no alterations in the expression of the essential NMDAR
subunit NR1, nor of two other NMDAR-associated MAGUKs, PSD-93 and
SAP-102 (FIG. 3A). This indicated that altered expression of NMDARs
and their associated proteins was unlikely to explain reduced NMDA
toxicity in PSD-95 deficiency (FIGS. 1C,D). Therefore, I examined
the possibility that PSD-95 modulates NMDAR function. NMDA currents
were recorded using the whole-cell patch technique (16) (FIG. 3B).
PSD-95 deficiency had no effect on passive membrane properties,
including input resistance and membrane capacitance [Capacitance:
AS 55.0.+-.2.6 pF (n=18 ); SE 52.7.+-.3.2 pF (n=19); SH 48.1.+-.3.4
pF (n=17; ANOVA, F=1.29, p=0.28)]. Whole-cell currents elicited
with 3-300 .mu.M NMDA were also unaffected. Peak currents were AS:
2340.+-.255 pA (n=18); SE: 2630.+-.276 (n=19); SH: 2370.+-.223
(n=17) (FIG. 3B, inset; one-way ANOVA, F=0.43, p=0.65). NMDA
dose-response relationships also remained unchanged (FIG. 3B;
EC.sub.50 AS: 16.1.+-.0.8 .mu.M (n=7); SE: 15.5.+-.2.1 (n=6); SH:
15.9.+-.2.9; one-way ANOVA, F=0.02, p=0.98), as were NMDA current
density and desensitization (FIG. 3C).
[0096] To further examine the effect of PSD-95 binding on NMDAR
function, a 9 aa peptide, KLSSIESDV (SEQ ID NO: 1) corresponding to
the C-terminal domain of the NR2B subunit characterized by the tSXV
motif (6) was injected into the neurons. At 0.5 mM, this peptide
competitively inhibited the binding of PSD-95 to GST-NR2B fusion
proteins (6), and was therefore predicted to uncouple NMDARs from
PSD-95. Intracellular dialysis of 1 mM tSXV or control peptide,
CSKDTMEKSESL (SEQ ID NO: 3) (6) was achieved through patch pipettes
(3-5 M.OMEGA.) also containing the fluorescent tracer Lucifer
Yellow (LY). This had no effect on NMDA currents over 30 min
despite extensive dialysis of LY into the cell soma and dendrites
(FIG. 3D). Peak current amplitudes were tSXV: 2660.+-.257 pA (n=9),
control: 2540.+-.281 pA (n=10; t.sub.(17)=0.31, p=0.76).
[0097] The data is consistent with that obtained from recently
generated mutant mice expressing a truncated 40K PSD-95 protein
that exhibited enhanced LTP and impaired learning (17). Hippocampal
CA1 neurons in PSD-95 mutants exhibited no changes in NMDAR subunit
expression and stoichiometry, cell density, dendritic
cytoarchitecture, synaptic morphology, or NMDAR localization using
NR1 immunogold labeling of asymmetric synapses. NMDA currents,
including synaptic currents, were also unchanged (16). I also found
no effects of PSD-95 deficiency on NMDAR expression, on other NMDAR
associated MAGUKs, nor on NMDA-evoked currents. In addition, NMDAR
function gauged by measuring NMDA-evoked
.sup.45Ca.sup.2+-accumulation was unaffected. Thus, the
neuroprotective consequences of PSD-95 deficiency must be due to
events downstream from NMDAR activation, rather than to altered
NMDAR function.
[0098] The second PDZ domain of PSD-95 binds to the C-terminus of
NR2 subunits and to other intracellular proteins (8). Among these
is nNOS (18), an enzyme that catalyzes the production of nitric
oxide (NO), a short-lived signaling molecule that also mediates
Ca.sup.2+-dependent NMDA toxicity in cortical neurons (12).
Although never demonstrated experimentally, the NMDAR/PSD-95/nNOS
complex was postulated to account for the preferential production
of NO by NMDARs over other pathways (8). To determine whether NO
signaling plays a role in NMDA toxicity in the present cultures, we
treated the cells with N.sup.G-nitro-L-arginine methyl ester
(L-NAME), a NOS inhibitor (12). L-NAME protected the neurons
against NMDA toxicity (FIG. 4A), indicating the possibility that
suppressing PSD-95 might perturb this toxic signaling pathway.
[0099] The effect of suppressing PSD-95 expression on NO signaling
and toxicity was examined using cGMP formation as a surrogate
measure of NO production by Ca.sup.2+-activated nNOS (20,21).
PSD-95 deficiency had no impact on nNOS expression (FIG. 4B), nor
on the morphology (FIG. 4B) or counts of NADPH diaphorase-staining
(12) neurons (SH: 361.+-.60, SE: 354.+-.54, AS: 332.+-.42 staining
neurons/10 mm coverslip, 3 coverslips/group). However, in neurons
lacking PSD-95 challenged with NMDA under conditions that isolated
Ca.sup.2+ influx to NMDARs (4), cGMP production was markedly
attenuated (>60%; FIG. 4C, one-way ANOVA, p<0.0001). Like
inhibited toxicity (FIGS. 1,2), inhibited cGMP formation in neurons
lacking PSD-95 was only observed in response to NMDA. It was
unaffected in neurons loaded with Ca.sup.2+ through VSCCs (FIG.
4D), even under high neuronal Ca.sup.2+ loads matching those
attained by activating NMDARs (compare FIGS. 1E and 2B2) (4). nNOS
function therefore, was unaffected by PSD-95 deficiency.
AMPA/kainate receptor activation failed to load the cells with
Ca.sup.2+ (FIG. 2A2), and thus failed to increase cGMP levels (FIG.
4E). Our findings indicate that suppressing PSD-95 selectively
reduces NO production efficiency by NMDAR-mediated Ca.sup.2+
influx, but preserves NO production by Ca.sup.2+ influx through
other pathways.
[0100] Bypassing nNOS activation with NO donors restored toxicity
in neurons lacking PSD-95. The NO donors sodium nitroprosside (12)
(FIG. 4F; EC.sub.50 300 .mu.M) and S-nitrosocysteine (17) (not
shown) were highly toxic, irrespective of PSD-95 deficiency. Thus,
reduced NMDA toxicity in PSD-95 deficient cells was unlikely to be
caused by altered signaling events downstream from NO
formation.
[0101] Suppressing PSD-95 expression uncoupled NO formation from
NMDAR activation (FIG. 4C), and protected neurons against NMDAR
toxicity (FIGS. 1C,D) without affecting receptor function (FIGS.
1E, 3A-D), by mechanisms downstream from NMDAR activation, and
upstream from NO-mediated toxic events (FIG. 4F). Therefore, PSD-95
imparts NMDARs with signaling and neurotoxic specificity through
the coupling of receptor activity to critical second messenger
pathways. These results have broader consequences, as NMDAR
activation and NO signaling are also critical to neuronal
plasticity, learning, memory, and behavior (1,18,19). Thus, these
data provide experimental evidence for a mechanism by which PSD-95
protein may govern important physiological and pathological aspects
of neuronal functioning.
[0102] FIG. 5 shows the utility of Tat-peptides in dissociating the
NMDAR/PSD-95 interaction (A) The hypothesis: The NMDAR/PSD-95
complex (left panel) may be dissociated using Tat peptides fused
either to the C-terminus of NR2B (Tat-NR2B9c; middle) or to the
first and second PDZ domains of PSD-95 (pTat-PDZ1-2; right), thus
reducing the efficiency of excitotoxic signaling via
Ca.sup.2+-dependent signaling molecules (B) Intracellular
accumulation of Tat-NR2B9c-dansyl (10 .mu.M) but not control
peptide (Tat-38-48-dansyl; 10 .mu.M) was observed 30 min after
application to cortical neuronal cultures using confocal microscopy
(excitation: 360 nm, emission: >510 nm; representative of 5
experiments). Fluorescence of cultures treated with
Tat-38-48-dansyl was similar to background (not shown). (C) Time
course of Tat-NR2B9c-dansyl (10 .mu.M) fluorescence after
application to cortical cultures at room temperature (symbols:
mean.+-.S.E of 4 experiments). Inset: fluorescence images from
representative experiment (D) Tat-NR2B9c, but not control peptides
(see text), inhibits the co-immunoprecipitation of PSD-95 with NR2B
in rat forebrain lysates (Left: Representative gel; Right:
means.+-.S.E of 4 experiments, ANOVA, F=6.086, *p=0.0041).
[0103] In more detail, a conserved tSXV motif at the C-terminus of
the NR2B subunit is critical for binding to the PDZ2 domain of
PSD-95. I hypothesized that interfering with this interaction might
disrupt the coupling between NMDARs and PSD-95. This might be
achieved by the intracellular introduction of exogenous peptides
that bind to either the NR2B or the PDZ2 interaction domains (FIG.
5A). To this end I used a peptide comprised of the nine C-terminal
residues of NR2B (KLSSIESDV; NR2B9c (SEQ ID NO: 1)), which is
anticipated to bind the PDZ2 domain of PSD-95. As an alternative
means to interfere with the NMDAR/PSD-95 interaction I constructed
a protein comprised of residues 65-248 of PSD-95 encompassing the
first and second PDZ domains (PDZ1-2), which contains the principal
binding domain in PSD-95 for the C-terminus of NR2B. NR2B9c or
PDZ1-2 on their own did not enter cells (not shown) and therefore,
I fused each to a peptide corresponding to the cell-membrane
transduction domain of the HIV-1-Tat protein (YGRKKRRQRRR (SEQ ID
NO: 2); Tat) to obtain a 20 amino acid peptide (Tat-NR2B9c) and the
fusion protein pTat-PDZ1-2. pTat-PDZ1-2 and pTat-GK fusion proteins
were generated by insertion of PSD95 residues 65-248 encoding the
PDZ 1 and 2, and residues 534-724 encoding the guanylate
kinase-like domains, respectively, into pTAT-HA plasmids (generous
gift of S. Dowdy, Washington University, St.Louis, Mo.). Fusion
proteins contain a 6X His-tag, the protein transduction domain of
HIV-1 Tat and a hemagglutinin-tag N-terminal to the insert.
Plasmids were transformed into BL21(DE3)LysS bacteria (Invitrogen)
and recombinant proteins were isolated under denaturing conditions
on a Nickle-His column (Amersham-Pharmacia). These are anticipated
to transduce cell membranes in a rapid, dose-dependent manner
independent of receptors and transporters (30).
[0104] To determine whether Tat-NR2B9c was able to transduce into
neurons, I conjugated the fluorophore dansyl chloride to Tat-NR2B9c
and to a control peptide comprised of HIV-1-Tat residues 38-48
(KALGISYGRKK (SEQ ID NO: 4); Tat38-48) outside the Tat transduction
domain (31).
[0105] Electrophysiological Recordings were made in 400 .mu.m
Hippocampal slices from 20-36 day old Sprague-Dawley rats perfused
at room temperature with ACSF containing (in mM) 126 NaCl, 3 KCl, 2
MgCl.sub.2, 2 CaCl.sub.2, 1.2 KH.sub.2PO.sub.4, 26 NaHCO.sub.3 and
10 glucose and bubbled with 95%O.sub.2/5%CO.sub.2. Whole-cell
recordings of CA1 neurons were performed using the "blind" method
with an Axopatch-1D amplifier (Axon Instruments, Foster City,
Calif.) at holding potential -60 mV. Pipettes (4-5 M.OMEGA.) were
filled with solution containing (mM): 135 CsCl, 2 MgCl.sub.2, 0.1
CaCl.sub.2, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.2 GTP, and 5 QX-314, pH
7.4, 310 mOsm. Field potentials were recorded with glass
micropipettes (2-4 M.OMEGA.) filled with ACSF placed in the stratum
radiatum 60-80 .mu.m from the cell body layer. Synaptic responses
were evoked by stimulation (0.05 ms) of the Schaffer
collateral-commissural pathway with a bipolar tungsten electrode in
the presence of bicuculline methiodide (10 .mu.M). For I.sub.NMDA
recording, Mg.sup.2+ was removed from and 20 .mu.M CNQX was added
in ACSF. Following 10-20 min base line recordings of EPSCs,
I.sub.NMDA and fEPSPs, Tat-peptides were applied in ACSF and
recordings were continued for 30 min thereafter.
[0106] I bath applied these to cultured cortical neurons and
observed their fluorescence by confocal microscopy. Neurons treated
with Tat-NR2B9c-dansyl (10 .mu.M) exhibited fluorescence in their
cytoplasm and processes, indicating intracellular peptide delivery
(FIG. 5B, left). Sister cultures treated with Tat38-48-dansyl (10
.mu.M) exhibited only background fluorescence, indicating no
observable peptide uptake in the absence of the Tat transduction
domain (FIG. 5B, right). Tat-NR2B9c-dansyl was detectable in the
neurons within 10 min of the start of the application and the
peptide accumulated to a maximum level over the next 20 min (FIG.
5C). This level was maintained until the dansyl-Tat-NR2B9c was
washed from the bath and the peptide remained detectable within the
neurons for more than 5 hours thereafter. Therefore, the Tat
transduction domain was able to act as a carrier for NR2B9c and the
Tat-NR2B9c fusion peptide remained in neurons for many hours after
being applied extracellularly.
[0107] To determine whether Tat-NR2B9c may disrupt the interaction
between NMDARs and PSD-95 I made use of rat brain proteins prepared
under weakly denaturing conditions known to permit the NMDAR/PSD-95
interaction. Adult (7-8W) wistar rat forebrains were removed and
homogenized in ice-cold buffer (0.32M Sucrose, 0.1 mM Na3VO4, 0.1
mM PMSF, 0.02M PNPP, 0.02M glycerol phosphate, and 5 ug/ml each of
antipain, aprotinin, and leupeptin). Homogenates were centrifuged
at 800 gr for 10 min at 4.degree. C. The supernatants were combined
and centrifuged at 11,000 g at 4 degree for 20 min and the pellet
(P2) was resuspended in homogenization buffer. P2 membranes were
adjusted 200 ug protein/90 ul with homogenization buffer with a
final concentration of 1% DOC and 0.1% Triton X-100. The proteins
were incubated with Tat-NR2B9c or with one of three controls:
Tat38-48, the Tat transduction sequence conjugated to two alanine
residues (Tat-AA), or a Tat-NR2B9c peptide in which the C-terminal
tSXV motif contained a double point mutation(Tat-KLSSIEADA;
Tat-NR2BAA) rendering it incapable of binding PSD-95. I
immunoprecipated NMDARs, together with associated proteins, with an
antibody that selectively recognizes NR2B. The proteins were
separated by SDS-PAGE and probed with anti-PSD-95 or anti-NR2B
antibodies.sup.16 NR2B was precipitated from rat forebrain extracts
using a polyclonal rabbit anti-NR2B antibody generated against the
C-terminal region encompassing amino acid residues 935-1,455 of the
NR2B protein. Proteins were then separated on 8% SDS-PAGE gels and
probed with monoclonal anti-NR2B (Clone 13, Transduction
Laboratories) or anti PSD-95 antibodies (Clone 7E3-1B8, Affinity
Bioreagents. Inc). Detection of proteins was achieved using
HRP-conjugated secondary antibodies and enhanced chemiluminescence.
I found that Tat-NR2B9c reduced the co-immunoprecipitation of
PSD-95 with NR2B. On average the optical density signal was reduced
by 37.6.+-.8.2% as compared with controls (FIG. 5D). In contrast,
none of the three control peptides reduced the
co-immmunoprecipitation of PSD-95 with NR2B. Thus, I conclude that
Tat-NR2B9c disrupts the interaction between NMDARs and PSD-95 and
that this is dependent upon an intact PDZ binding motif in the
peptide.
[0108] FIG. 6 shows neuroprotection and reduction of NO signaling
by Tat-peptides without affecting NMDAR function (A) Effect of
Tat-NR2B9c (50 nM) on field excitatory post-synaptic currents
(fEPSC) in CA1 neurons in acute hippocampal slices. (B) Effect of
50 nM Tat-NR2B9c or Tat-38-48 (control) on whole-cell excitatory
post synaptic currents (EPSC). (C) Effect of Tat-NR2B9c on the NMDA
component of the EPSC isolated pharmacologically by applying the
AMPAR antagonist CNQX, and concomitant removal of extracellular
Mg.sup.2+. (D) Effect of 50 nM Tat-NR2B9c treatment on NMDA-evoked
.sup.45Ca.sup.2+ uptake in cortical cultures. Tat-peptides were
bath-applied 1 h prior to the NMDA application. (E) Effect of 50 nM
Tat-NR2B9c treatment on NMDA-evoked cGMP production in cortical
cultures. Asterisk: differences from control and Tat-NR2B-AA at
each NMDA concentration (Bonferroni t-test, p<0.01). (F)
Decreased excitotoxicity at 20 h at all NMDA concentrations in
cultured cortical neurons pre-treated with 50 nM Tat-NR2B9c or
pTat-PDZ1-2 for 1 h. Asterisk: differences from control,
Tat-NR2B-AA and pTat-GK at each NMDA concentration (Bonferroni
t-test, p<0.005). Right panels: Representative phase contrast
and propodium iodide fluorescence images of cultures 20 h after
challenge with 100 .mu.M NMDA with and without Tat-NR2B9c
treatment. Bars in (D), (E) and (F) indicate the mean.+-.S.E. for
12 cultures in 3 separate experiments.
[0109] In more detail, as NMDAR-mediated synaptic responses are not
altered by the loss of PSD-95 (24) I predicted that Tat-NR2B9c
would not affect the function of NMDARs. This was tested by
examining the effect of Tat-NR2B9c on NMDAR-mediated currents and
on NMDA-evoked uptake of .sup.45Ca.sup.2+. Bath-applying Tat-NR2B9c
(50 nM) to acute rat hippocampal slices had no effect on synaptic
responses of CA1 neurons evoked by stimulation of the Schaffer
collateral-commissural pathway (FIG. 6A) nor on patch recordings of
the total excitatory post-synaptic currents (EPSC) recorded in CA1
neurons, (FIG. 6B) nor on the pharmacologically isolated AMPA (not
shown) or NMDA components of the EPSC (FIG. 6C). Moreover, using
cortical cultures I found that pre-treating cultures with
Tat-NR2B9c or with pTat-PDZ1-2 (each at 50 nM) did not alter the
uptake of .sup.45Ca.sup.2+ produced by applying NMDA (FIG. 6D);
CNQX (10 .mu.M) and nimodipine (2 .mu.M) were present in the
extracellular solution in these and all subsequent experiments
using cultured neurons so as to isolate signaling and thereby
preventing secondary activation of AMPARs or of voltage-gated
Ca.sup.2+ channels, respectively (25,32,33).
[0110] As the function of NMDARs was unaffected by administering
Tat-NR2B9c, I next determined whether this peptide altered
signaling events downstream of NMDAR activation. To this end I
examined stimulation of nNOS, as a key downstream signaling enzyme
that mediates the neurotoxic effects of NMDAR activation.sup.5. I
measured NMDA-evoked changes in the levels of guanosine
3',5'-monophosphate (cGMP) as a surrogate measure of NO production
by NMDAR stimulated nNOS activity.sup.7,20. Cultured cortical
neurons were pre-treated for 1 h with Tat-NR2B9c (50 nM), the
non-interacting Tat-NR2B-AA (50 nM) or with sham washes and
challenged with NMDA (0-1000 .mu.M) in the presence of CNQX and
nimodipine as above. NMDA produced a concentration-dependent
increase in cGMP that was significantly suppressed (average of
39.5.+-.6.7%) by pre-treating the cultures with Tat-NR2B9c (FIG.
6E). In contrast, NMDAR-stimulated elevation of cGMP was unaffected
by pre-treatment with Tat-NR2B-AA. Thus, Tat-NR2B9c, but not a
mutant peptide incapable of interacting with PSD-95, depressed
NMDAR-evoked stimulation of NO-cGMP signaling.
[0111] Although Tat-NR2B9c and pTat-PDZ1-2 did not affect NMDAR
function, Tat-NR2B9c was shown to interfere with NMDAR/PSD-95
binding and to suppress downstream NO signaling. Thus, I predicted
that Tat-peptide treatment should enhance neurons' resilience to
NMDA toxicity. To test this I pre-treated cortical neuronal
cultures with Tat-peptides (50 nM) for 1 h, then applied NMDA
(0-100 .mu.M) for 1 h followed by a 20 h observation period (FIG.
6F, inset). Control neurons were treated with sham washes, or with
the non-interacting control Tat-NR2BAA. In cultures treated with
Tat-NR2B9c, cell death was significantly reduced at all
concentrations tested (FIG. 6F) whereas pre-treatment with
Tat-NR2B-AA had no effect on cell death. Thus, NMDAR-stimulated
neurotoxicity is suppressed by pre-treatment with Tat-NR2B9c,
suppression that is lost by mutating the PSD-95 binding region of
the peptide.
[0112] If Tat-NR2B9c suppresses NMDA excitotoxicity by interfering
with the binding of NR2B to PSD-95 then interfering with this
binding by an alternative means should also suppress the toxicity.
I tested pTat-PDZ1-2, predicted to interfere with PSD-95 binding to
NR2B and which permeates into the cells (not shown), though without
effect on NMDA-evoked Ca.sup.2+ accumulation (FIG. 6D).
Pre-treating the cultures with pTat-PDZ1-2 attenuated the
neurotoxicity of NMDA to a similar degree as Tat-NR2B9c (FIG. 6F).
As a control, I made and used pTat-GK, a Tat fusion protein
containing residues 534-724 of PSD-95 comprising the
carboxyl-terminal guanylate-kinase homology domain that lacks
enzymatic activity.sup.21. pTat-GK, which is devoid of the
necessary domains to bind NR2B, had no effect on the NMDA-evoked
cell death (FIG. 6F). Thus, interfering with the NMDAR/PSD-95
interaction using peptides that target either side of the
interaction reduces in vitro excitotoxicity produced by NMDAR
activation.
[0113] FIG. 7 shows neuroprotection by Tat-NR2B9c pretreatment
in-vivo. (A) Detection of Tat-NR2B9c-dansyl but not Tat38-48-dansyl
in the cortex of C57BL/6 mouse brain 1 h after intraperitoneal
injection (0.5 .mu.mole total dose). Fluorescence of brains from
animals treated with Tat-38-48-dansyl was similar to background
(not shown). (B) Composite neurological scores (see text) during
and 24 h after MCAo. (C) Pre-treatment with 3 nmole/g Tat-NR2B9c
but not mutated Tat-NR2B-AA or saline (control) significantly
reduced (i) total infarct area and volume (inset), ANOVA; F=7.3,
p<0.005 and (ii) cortical infarct area and volume (inset),
ANOVA; F=8.35, p<0.005 measured 24 h after transient MCAo. (n=6
animals per group; symbols and bars indicate mean.+-.S.E). Infarct
volume was calculated by analyzing the infarct area in 8
stereotactic coordinates of the brain as shown at right inset.
[0114] Agents that block NMDAR activity were initially deemed as
promising neuroprotectants for stroke and other neurological
disorders involving excitotoxic mechanisms, but were later shown to
be deleterious or ineffective in animal and human studies
(27,28,29). However, Tat-peptides that target the NMDAR/PSD-95
interaction protect against NMDA toxicity without blocking NMDARs.
Therefore I reasoned that treatment with Tat-NR2B9c in vivo could
serve as an improvement on NMDA blockers in the treatment of
ischemic brain damage.
[0115] Before testing this I determined whether Tat-NR2B9c could be
delivered into the brain in the intact animal. I injected 25 g
C57BL/6 mice intraperitoneally with a 500 .mu.mole dose of either
Tat-NR2B9c-dansyl, or with Tat38-48-dansyl as a non-transducing
control. 40 .mu.m cryostat coronal brain sections taken 1 h after
injection.sup.22 were examined for peptide uptake using dansyl
fluorescence detection by confocal microscopy. The mice were
perfused with fixative solution (3% paraformaldehyde, 0.25%
glutaraldehyde, 10% sucrose, 10 U/mL heparin in Saline) 1 hour
after peptide injection. Brains were removed, frozen in
2-methylbutane at -42.degree. C. and 40.quadrature. m sections were
cut using a Leitz Kryostat. Brain sections from animals injected
with Tat-NR2B9c exhibited strong fluorescence in the cortex (FIG.
7A, right), and in all other areas examined (hippocampus, striatum;
not shown), whereas signal from controls remained at background
levels (FIG. 7A, left). Similar results were obtained using
intravenous injection in rats (not shown). Thus, Tat-NR2B9c enters
the brain upon peripheral administration.
[0116] Next, I examined whether pretreatment with Tat-peptides
would reduce stroke damage. Experiments were carried out in adult
male Sprague-Dawley rats subjected to transient middle cerebral
artery occlusion (MCAO) for 90 minutes by the intraluminal suture
method (36,37). Animals were fasted overnight and injected with
atropine sulfate (0.5 mg/kg IP). After 10 minutes anesthesia was
induced with 3.5% halothane in a mixture of nitrous oxide and
oxygen (Vol. 2:1) and maintained with 0.8% halothane. Rats were
orally intubated, mechanically ventilated, and paralyzed with
pancuronium bromide (0.6 mg/kg IV). Body temperature was maintained
at 36.5-37.5.degree. C. with a heating lamp. Polyethylene catheters
in the femoral artery and vein were used to continuously record
blood pressure and to sample blood for gas and pH measurements.
Transient MCAO was achieved for 90 min by introducing a
poly-L-lysine-coated 3-0 monofilament nylon suture (Harvard
Apparatus) into the circle of Willis via the internal carotid
artery, effectively occluding the middle cerebral artery. This
produces an extensive infarction encompassing the cerebral cortex
and basal ganglia. Animals were pretreated with either saline, the
Tat-NR2B-AA control, or with Tat-NR2B9c by a single intravenous
bolus injection 45 min prior to MCAO (3 nMoles/g). Physiological
parameters (body temperature, blood pressure, blood gases) were
monitored and maintained throughout the experiment (Table 1). All
experimental manipulations and analyses of data were performed by
individuals blinded to the treatment groups. The extent of cerebral
infarction was measured 24 h after MCAO onset (FIG. 7C inset). The
postural reflex test (38), and the forelimb placing test (39) were
used to grade neurological function on a scale of 0 to 12
(normal=0; worst=12) during MCAO (at 50 minutes) and 24 h
thereafter.
[0117] Pretreatment with Tat-NR2B9c produced a trend toward
improvement in 24 h neurological scores in animals treated with
Tat-NR2B9c (FIG. 7B). Moreover, the treatment reduced the volume of
total cerebral infarction by 54.6.+-.11.27% as compared with stroke
volume in controls (FIG. 7C.sub.i; ANOVA, F=7.289, p=0.0048). This
effect was largely accounted-for by a 70.7.+-.11.23% reduction in
cortical infarction (FIG. 7C.sub.ii, ANOVA, F=8.354, p=0.0027),
which is thought to be largely caused by NMDAR-dependent
mechanisms.
[0118] A treatment for stroke with a single-bolus drug injection
would be most therapeutically valuable if effective when given
after the onset of ischemia. I thus first evaluated whether
treatment with Tat-peptides could be neuroprotective when applied
post-insult in vitro.
[0119] FIG. 8 shows neuroprotection by post-treatment with
Tat-NR2B9c in-vitro and in-vivo (A) Decreased excitotoxicity at 20
h in cultured cortical neurons post-treated with 50 nM Tat-NR2B9c
or pTat-PDZ1-2 at 1 h after NMDA application. Bars indicate the
mean.+-.S.E. for 12 cultures in 3 separate experiments. Asterisk:
differences from control, Tat-NR2B-AA and pTat-GK at each NMDA
concentration (Bonferroni t-test, p<0.005). Right panels:
Representative phase contrast and propodium iodide fluorescence
images of cultures 24 h after challenge with 100 .mu.M NMDA with
and without Tat-NR2B9c treatment. (B) Composite neurological scores
(see text) during and 24 h after MCAo. Asterisk: difference from
control and Tat-NR2B-AA (ANOVA; F=17.25, p<0.0001). (C)
Post-treatment with 3 nmole/g Tat-NR2B9c (9 animals) but not
mutated Tat-NR2B-AA (8 animals) or saline controls (10 rats)
significantly reduced (i) total infarct area and volume (inset),
ANOVA; F=12.0, p<0.0005 and (ii) cortical infarct area and
volume (inset), ANOVA; F=12.64, p=0.0001 as measured 24 h after
transient MCAo. Symbols and bars indicate mean.+-.S.E (D).
Representative appearance of H&E stained rat brain sections
from which the infarct areas were analyzed.
[0120] Cultured cortical neurons were exposed to an NMDA challenge
(0-100 .mu.M) for 1 h and were then treated with the Tat-peptides
(all at 50 nM) described in the pre-treatment study (FIG. 6F). Cell
death was gauged 20 h thereafter (FIG. 8A--inset). Post-treatment
with Tat-NR2B9c or with pTat-PDZ1-2 significantly reduced the
vulnerability of neurons to NMDA toxicity as compared with control
cultures post-treated with sham washes, with Tat-NR2BAA, or with
pTat-GK (FIG. 8A). Thus, when administered 1 hr after the start of
the NMDA insult each of the Tat fusion constructs that target the
NMDAR/PSD-95 interaction significantly reduced neuronal cell death
in vitro.
[0121] Finally, I examined whether treatment with Tat-NR2B9c could
attenuate ischemic neuronal damage in-vivo when given after stroke
onset. A post-treatment study was conducted in which the rats were
subjected to transient MCAO for 90 minutes as before, but the
intravenous saline or Tat-peptide bolus (Tat-NR2B9c or Tat-NR2B-AA;
3 nMole/g) was injected 1 h after MCAO onset (FIG. 8C--inset).
Infarction volume and neurological outcome measurements were
performed at times identical to the pre-treatment study. Body
temperature, blood pressure and blood gases were monitored
throughout the 24 h experiment and maintained equivalent between
groups (Table 2).
[0122] Post-treatment with Tat-NR2B9c, but not with Tat-NR2B-AA or
saline, resulted in animals exhibiting a significant improvement in
24 h neurological scores as compared with controls (FIG. 8B; ANOVA,
F=17.25, p<0.0001). Most strikingly, post-treatment with
Tat-NR2B9c reduced the volume of total cerebral infarction by
67.0.+-.3.75% as compared with stroke volume in controls (FIG.
8C.sub.i; ANOVA, F=11.99, p=0.0002). Similar to the previous study,
this reduction was accounted-for by a 86.97.+-.4.38% reduction in
cortical infarction volume (FIG. 8C.sub.ii, 4D; ANOVA, F=12.64,
p<0.0001).
[0123] The aforesaid description demonstrates that introducing into
cells an exogenous peptide containing the C-terminal nine amino
acids of the NR2B NMDAR subunit has profound effects on signaling
pathways downstream of NMDAR activation, on in vitro
excitotoxicity, and on in vivo ischemic brain damage. The effects
of this peptide are lost by mutating amino acids that are essential
for mediating PDZ binding to PSD-95. In addition, a protein
comprising PDZ1-2 of PSD-95 shares the effects of the NR2B
C-terminal peptide. Together these findings imply that the
downstream signaling from NMDARs that leads to negative
consequences for neuronal viability may be interrupted by
interfering with the interaction between NR2B and PSD-95.
[0124] I have discovered that the strategy of treating neurons with
Tat-fusion peptides is effective in reducing vulnerability to
excitotoxicity in vitro and stroke damage in vivo. As this occurs
without affecting NMDAR activity then adverse consequences of
blocking NMDARs are not expected. Efficacy after the insult onset
suggests that targeting the NMDAR/PSD-95 interaction is a practical
future strategy for treating stroke. It is also likely that
targeting other intracellular proteins using the same approach
could be used to modulate additional signaling mechanisms mediated
by protein-protein interactions that lead to other human
diseases.
1TABLE 1 Physiological Variables in Pre-Treatment MCAO Study
Control TAT-NR2BAA TAT-NR2B9c Physiological Variables (n = 6) (n =
6) (n = 6) Before anesthesia Body weight, g 269 .+-. 6 273 .+-. 7
271 .+-. 5 Before MCAo (45 min) Body Temperature, .degree. C. 36.7
.+-. 0.07 36.7 .+-. 0.17 36.6 .+-. 0.21 MABP, mmHg 119 .+-. 4 115
.+-. 5 120 .+-. 9 Before MCAo (30 min) Body Temperature, .degree.
C. 36.8 .+-. 0.08 36.5 .+-. 0.12 36.7 .+-. 0.19 MABP, mmHg 107 .+-.
3 110 .+-. 4 76 .+-. 5* Blood gases PH 7.44 .+-. 0.02 7.44 .+-.
0.02 7.44 .+-. 0.02 PO2, mmHg 104 .+-. 3 110 .+-. 7 123 .+-. 8
PCO2, mmHg 39.6 .+-. 1.3 39.1 .+-. 1.4 38.1 .+-. 1.4 Before MCAo
(15 min) Body Temperature, .degree. C. 36.9 .+-. 0.11 36.6 .+-.
0.15 36.7 .+-. 0.20 MABP, mmHg 111 .+-. 6 115 .+-. 5 90 .+-. 6*
During MCAo (5 min) Body Temperature, .degree. C. 36.9 .+-. 0.03
36.6 .+-. 0.17 36.7 .+-. 0.16 MABP, mmHg 132 .+-. 6 135 .+-. 7 112
.+-. 9 Blood gases PH 7.44 .+-. 0.02 7.44 .+-. 0.02 7.44 .+-. 0.02
PO2, mmHg 118 .+-. 3 109 .+-. 4 112 .+-. 6 PCO2, mmHg 39.2 .+-. 0.6
39.6 .+-. 0.5 41.0 .+-. 1.3 During MCAo (15 min) Body Temperature,
.degree. C. 36.9 .+-. 0.09 36.7 .+-. 0.15 36.8 .+-. 0.23 MABP, mmHg
116 .+-. 9 111 .+-. 6 98 .+-. 6 After MCAo (15 min) Body
Temperature, .degree. C. 36.9 .+-. 0.09 36.8 .+-. 0.08 36.8 .+-.
0.12 After MCAo (24 hr) Body Temperature, .degree. C. 36.6 .+-.
0.14 37.0 .+-. 0.25 36.5 .+-. 0.14 Body weight, g 238 .+-. 6 244
.+-. 6 250 .+-. 5 MABP: Mean arterial blood pressure *P < 0.05,
Student's t-test
[0125]
2TABLE 2 Physiological Variables in Post- Treatment MCAO Study
Control TAT-NR2BAA TAT-NR2B9c Physiological Variables (n = 10) (n =
8) (n = 9) Before anesthesia Body weight, g 314 .+-. 4 301 .+-. 5
306 .+-. 7 Before MCAo (15 min) Body Temperature, .degree. C. 36.9
.+-. 0.07 36.7 .+-. 0.07 36.6 .+-. 0.07 MABP, mmHg 103 .+-. 4 103
.+-. 6 103 .+-. 5 Blood gases PH 7.43 .+-. 0.01 7.45 .+-. 0.01 7.43
.+-. 0.02 PO2, mmHg 113 .+-. 4 113 .+-. 4 105 .+-. 4 PCO2, mmHg
39.4 .+-. 1.0 37.9 .+-. 1.1 40.1 .+-. 1.0 During MCAo (15 min) Body
Temperature, .degree. C. 36.9 .+-. 0.07 36.7 .+-. 0.11 37.0 .+-.
0.07 MABP, mmHg 120 .+-. 5 121 .+-. 5 119 .+-. 8 Blood gases PH
7.44 .+-. 0.01 7.46 .+-. 0.01 7.43 .+-. 0.01 PO2, mmHg 113 .+-. 3
108 .+-. 2 111 .+-. 4 PCO2, mmHg 39.3 .+-. 0.7 48.0 .+-. 1.2 39.8
.+-. 0.9 During MCAo (60 min) Body Temperature, .degree. C. 37.1
.+-. 0.21 37.0 .+-. 0.31 36.7 .+-. 0.11 MABP, mmHg 146 .+-. 5 149
.+-. 4 143 .+-. 5 During MCAo (65 min) Body Temperature, .degree.
C. 37.1 .+-. 0.16 37.0 .+-. 0.29 36.9 .+-. 0.08 MABP, mmHg 134 .+-.
6 136 .+-. 5 137 .+-. 4 After MCAo (15 min) Body Temperature,
.degree. C. 37.0 .+-. 0.09 36.9 .+-. 0.23 36.8 .+-. 0.08 MABP, mmHg
128 .+-. 6 116 .+-. 4 119 .+-. 4 After MCAo (24 hr) Body
Temperature, .degree. C. 36.6 .+-. 0.14 36.7 .+-. 0.27 36.4 .+-.
0.24 Body weight, g 276 .+-. 3 276 .+-. 6 279 .+-. 8 MCAo: Middle
cerebral artery occlusion; MABP: Mean arterial blood pressure
[0126] Although this disclosure has described and illustrated
certain preferred embodiments of the invention, it is to be
understood that the invention is not restricted to those particular
embodiments. Rather, the invention includes all embodiments which
are functional or mechanical equivalence of the specific
embodiments and features that have been described and
illustrated.
* * * * *